The first event in light perception is absorption of a photon by an opsin pigment molecule, which causes isomerization of the 11-cis-retinaldehyde chromophore. Before light sensitivity can be restored, the resulting all-tans-retinaldehyde must be chemically re-isomerized to 11-cis-retinaldehyde by an enzymatic process called the visual cycle. Most steps of the visual cycle take place within the retinal pigment epithelium (RPE), a layer of cells adjacent to the photoreceptor outer-segments. Several enzymes of the visual cycle have not been well characterized and their genes not yet cloned. One such enzyme is isomerohydrolase, which catalyzes the critical all-trans to 11-cis re-isomerization steps. Another is all-trans-retinyl ester hydrolase, which hydrolyzes all trans-retinyl esters to yield all-trans-retinol and a fatty acid. Still another is 11-cis-retinyl ester hydrolase, which similarly hydrolyzes 11-cis-retinyl esters. Although these hydrolase activities have been well documented in RPE membranes, the functions of all-trans-retinyl ester hydrolase and 11-cis-retinyl ester hydrolase in the visual cycle are not well understood. The first Specific Aim of this proposal is to purify isomerohydrolase, all-trans-retinyl ester hydrolase, and 11-cis-retinyl ester hydrolase. The mRNA's and genes for these enzymes will be cloned using two complementary strategies. The function of each protein in the visual cycle will be studied by generating 'knockout' mice with a null mutation in the gene. Also, the gene for each protein will be evaluated as a cause of inherited retinal dystrophy in humans. If disease-causing alleles are found, the biochemical mechanisms will be studied using transgenic and knockout mice. Virtually nothing is known about regulation of the visual cycle. Two recent observations in our laboratory on rpe65-/- knockout mice and cultured RPE cells showed light-mediated mobilization of retinyl esters. These observations indicate that RPE cells are intrinsically sensitive to light. Further, these observations imply the existence of a novel-signaling pathway in RPE cells that regulates one or more steps in the visual cycle. Also, we have tentatively identified the light-receptor molecule for this regulatory pathway. Specific Aims II and III of this proposal are to characterize this regulatory pathway biochemically and genetically, using tissue culture and mouse genetic models. The goals of this section are to identify the opsin photopigment for this regulatory pathway that confers light-sensitivity, to define the G protein alpha-subunit, and to determine the catalytic step(s) in the visual cycle regulated by this pathway.